To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P Ͻ 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin-and AMPKactivated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P Ͻ 0.05). AMPK-and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P Ͻ 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P Ͻ 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.glucose transporter 4; mitogen-activated protein kinase; phosphatidylinositol 3-kinase; enhanced green fluorescent protein IT HAS BEEN ESTABLISHED that insulin-stimulated glucose uptake into adipocytes and skeletal myocytes involves the translocation of GLUT4 from an intracellular pool to the plasma membrane. The intracellular mechanism for the recruitment of GLUT4-containing vesicle into plasma membrane has been investigated, and it has been revealed that phosphatidylinositol 3-kinase (PI3K) plays a crucial role in insulin-stimulated GLUT4 translocation (7,14,24). However, little has been elucidated concerning other mechanisms to enhance the GLUT4 translocation than the insulin signaling system. AMP-activated protein kinases (AMPKs) have been known to act as a metabolic sensor in mammalian cells (9,30). The kinase activity is enhanced by a relative increase in cellular AMP level (increase in AMP-to-ATP ratio) through a metabolic uncoupler, dinitrophenol (DNP), to decrease ATP concentration and by 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (AICAR), an adenosine analog. After entering into cells, AICAR is phosphorylated, and ...

H. JNK-and IB-dependent pathways regulate MCP-1 but not adiponectin release from artificially hypertrophied 3T3-L1 adipocytes preloaded with palmitate in vitro. Am J Physiol Endocrinol Metab 294: E898-E909, 2008. First published February 26, 2008 doi:10.1152/ajpendo.00131.2007.-Obese conditions increase the expression of adipocytokine monocyte chemoattractant protein-1 (MCP-1) in adipose tissue as well as MCP-1 plasma levels. To investigate the mechanism behind increased MCP-1, we used a model in which 3T3-L1 adipocytes were artificially hypertrophied by preloading with palmitate in vitro. As observed in obesity, under our model conditions, palmitate-preloaded cells showed significantly increased oxidative stress and increased MCP-1 expression relative to control cells. This increased MCP-1 expression was enhanced by adding exogenous tumor necrosis factor-␣ (TNF-␣; 17.8-fold vs. control cells, P Ͻ 0.01) rather than interleukin-1␤ (IL-1␤; 2.6-fold vs. control cells, P Ͻ 0.01). However, endogenous TNF-␣ and IL-1␤ release was not affected in hypertrophied cells, suggesting that these endogenous cytokines do not mediate hypertrophy-induced increase in MCP-1. MCP-1 secretion from hypertrophied cells was significantly decreased by treatment with antioxidant N-acetyl-cysteine, JNK inhibitors SP600125 and JIP-1 peptide, and IB phosphorylation inhibitors BAY 11-7085 and BMS-345541 (P Ͻ 0.01). MCP-1 secretion was not affected by peroxisome proliferator-activated receptor-␥ (PPAR␥) antagonists assayed. Adiponectin, another adipocytokine studied in parallel, also showed increased release in hypertrophy relative to control cells. But in contrast to MCP-1, adiponectin release was significantly suppressed by both exogenous TNF-␣ and IL-1␤ as well as by PPAR␥ antagonists bisphenol A diglycidyl ether and T0070907 (P Ͻ 0.01). JNK inhibitors and IB phosphorylation inhibitors showed no significant effect on adiponectin. We conclude that adipocyte hypertrophy through palmitate loading causes oxidative stress, which in turn increases MCP-1 expression and secretion through JNK and IB signaling. In contrast, the parallel increase in adiponectin expression appears to be related to the PPAR␥ ligand properties of palmitate.c-Jun NH 2-terminal kinase; monocyte chemoattractant protein-1; tumor necrosis factor-␣; interleukin-1␤ ADIPOCYTES STOCKPILE TRIGLYCERIDES, and increasing adipocyte size or hypertrophy accompanies the increased intracellular triglyceride content (43). In addition to triglyceride stockpiling, adipocytes function in endocrine signaling by secreting adipocytokines such as adiponectin, tumor necrosis factor-␣ (TNF-␣), interleukin-1␤ (IL-1␤), and monocyte chemoattractant protein-1 (MCP-1) (31). Adipocytokine MCP-1 is a potent chemotactic factor for monocytes and is predominantly produced by macrophages and vascular endothelial cells (34). MCP-1 expression in adipose tissue leads to macrophage infiltration into this tissue and insulin resistance under obese conditions (5,17,18). In vivo in obese mice, MCP-1 is abundantly exp...

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